Compositions and methods for detecting Enterovirus D68

• US 9,938,588 B2
• Filed: 06/03/2016
• Issued: 04/10/2018
• Est. Priority Date: 06/05/2015
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Nix, William Allan, Oberste, M. Steven
• Primary Examiner(s): Gill, Rachel B
• Application Number:
  US15/173,450
• Publication Number:
  US 20160355897A1
• Time in Patent Office: 676 Days
• CPC Class Codes: A61K 2039/6075 Viral proteins
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2770/32311 Enterovirus
• CPC Class Codes: C12N 2770/32351 Methods of production or purification of viral material
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: G01N 33/56983 Viruses

Abstract

Methods and compositions for detection of enterovirus D in a sample, particularly detection of enterovirus D68, are provided. The methods include contacting a sample with at least one primer (such as a forward primer and/or a reverse primer) capable of specifically amplifying an EV-D68 viral protein 1 (VP1) nucleic acid or a portion thereof and/or a detectably labeled probe capable of specifically hybridizing to an EV-D68 VP1 nucleic acid, under conditions sufficient for specific amplification of the EV-D68 VP1 nucleic acid by the at least one primer and/or under conditions sufficient for specific hybridization of the probe to the EV-D68 nucleic acid. The amplification of the EV-D68 VP1 nucleic acid and/or the hybridization of the probe to the EV-D68 VP1 nucleic acid is detected, thereby identifying presence of EV-D68 in the sample.

Claims

1. A method of detecting presence of human enterovirus D68 (EV-D68) in a sample, comprising:

contacting the sample with;

a forward primer and a reverse primer capable of amplifying an EV-D68 viral protein 1 (VP1) nucleic acid or portion thereof, wherein the forward primer consists of the nucleic acid sequence of SEQ ID NO;

1 and the reverse primer consists of the nucleic acid sequence of SEQ ID NO;

2; and

a detectably labeled probe consisting of the nucleic acid sequence of SEQ ID NO;

3 and a detectable label,

under conditions sufficient for amplification of the EV-D68 VP1 nucleic acid by the forward and reverse primer and hybridization of the probe to the EV-D68 VP1 nucleic acid; and

detecting the amplification of the EV-D68 VP1 nucleic acid and/or hybridization of the probe to the EV-D68 VP1 nucleic acid, thereby detecting presence of human enterovirus D68 in the sample.

2. The method of claim 1, wherein the detectably labeled probe comprises at least one fluorophore.

3. The method of claim 2, wherein the detectably labeled probe comprises a donor fluorophore, an acceptor fluorophore, or a combination thereof.

4. The method of claim 3, wherein the detectably labeled probe comprises the donor fluorophore FAM or CY5 and the acceptor fluorophore BHQ1 or BHQ2.

5. The method of claim 4, wherein the detectably labeled probe consists of FAM-GTCCATTTGAAAAAGTTCTTGTC-BHQ1 (SEQ ID NO:

3) or CY5-GTCCATTTGAAAAAGTTCTTGTC-BHQ2 (SEQ ID NO;

     3).

6. The method of claim 1, wherein the sample comprises isolated DNA, isolated RNA, serum, a nasal wash, a respiratory aspirate, a nasopharyngeal swab, or an oropharyngeal swab.

7. An isolated probe consisting of the nucleic acid sequence of SEQ ID NO:

3 and a detectable label.

8. The isolated probe of claim 7, wherein the detectable label comprises one or more fluorophores, chromogenic moieties, haptens, affinity tags, or radioactive isotopes.

9. The isolated probe of claim 8, wherein the detectable label comprises a donor fluorophore, an acceptor fluorophore, or a combination thereof.

10. The isolated probe of claim 9, wherein the detectable label comprises the donor fluorophore FAM or CY5 and the acceptor fluorophore BHQ1 or BHQ2.

11. The isolated probe of claim 10, wherein the probe consists of FAM-GTCCATTTGAAAAAGTTCTTGTC-BHQ1 (SEQ ID NO:

3) or CY5-GTCCATTTGAAAAAGTTCTTGTC-BHQ2 (SEQ ID NO;

     3).

12. A kit for detecting human enterovirus D68 in a sample, comprising:

a detectably labeled probe consisting of the nucleic acid sequence of SEQ ID NO;

3 and a detectable label;

a forward primer consisting of the nucleic acid sequence of CAAACTCGCACAGTGATAAAYCARCA (SEQ ID NO;

     1); and

a reverse primer consisting of the nucleic acid sequence of GTATTATTACTACTACCATTCACNGCNAC (SEQ ID NO;

     2).

13. The kit of claim 12, wherein the detectably labeled probe consists of FAM-GTCCATTTGAAAAAGTTCTTGTC-BHQ1 (SEQ ID NO:

3) or CY5-GTCCATTTGAAAAAGTTCTTGTC-BHQ2 (SEQ ID NO;

     3).

14. The kit of claim 13, further comprising:

a detectably labeled probe capable of hybridizing to a human RNase P nucleic acid;

a forward primer capable of hybridizing to the human RNase P nucleic acid; and

a reverse primer capable of hybridizing to the human RNase P nucleic acid.